All vertebrates exhibit left-right (L-R) asymmetry in the positions of their visceral organs. The heart is the first organ to exhibit this asymmetry and as a consequence it invariantly loops to the right side. Disturbances in LR asymmetry underlie a variety of complex heart defects including heterotaxy, L-transposition of the Great Arteries (L-TGA) and dextocardia. X-linked Heterotaxy (HTX1) is caused by mutations in the ZIC3 locus. ZIC3 encodes a C2H2 class zinc-finger transcription homologous to Drosophila odd-paired. ZIC3 along with its paralogs ZIC1 and ZIC2 forms a superfamily with the GL1 transcription factors (cubitus-interruptus, GLI1, GLI2, and GLI3). Although great progress has made in unraveling the molecular networks controlling LR axis formation, the role of ZIC3 is unknown. Three major processes are apparently involved in establishing the LR axis: (1) establishment of asymmetric signaling from the node; (2) development of the midline boundary; and (3) asymmetric induction of the lateral plate mesoderm (LPM) by nodal and related pathways. The purpose of these studies is to investigate the mechanistic role of ZIC3 in one or more of these processes. We hypothesize that ZIC3 acts as a regulatory in the sonic hedgehog (Shh)-GLI pathway in producing midline structures and a LR boundary. The first specific aim will continue anatomic characterization of mice bearing a null mutation in the ZIC3 locus. Preliminary data indicates that this model exhibits disturbances in laterality right pulmonary isomerism and cardiac defects. We propose to use markers of midline and nodal pathway processes to investigate their possible dependence on ZIC3 function. The second specific aim will investigate possible genetic interactions with Shh and GLI. These studies are expected to address the potential functional alteration of Shh signaling by ZIC3. The third specific aim will focus on identification of ZIC3 transcriptional regulatory elements necessary to carry out transgenic expression studies. Specific aim 4 will examine the consequences of ZIC3 and mutant ZIC3 in the node and midline. These experiments will help to establish the mechanisms by which ZIC3 exerts its effect on LR asymmetry.